This invention relates to high level microbial production of bovine growth hormone through recombinant DNA technology. This high level production is achieved through high-density fermentation of E. coli cells transformed with a recombinant vector carrying a gene encoding bovine growth hormone.
Bovine growth hormone (BGH) is a protein of 191 amino acids, which is initially synthesized in the anterior pituitary land as a precursor "pre-growth hormone" having 26 additional amino acids attached at the N-terminus. This 26-amino acid "signal sequence" is processed off during secretion from the pituitary cells, yielding the mature hormone. Field trials using BGH purified from pituitary glands demonstrated increased milk production and improved feed-to-milk conversion in cows to which the hormone was administered (Machlin, L. J., Journal of Dairy Science, 56:575-580 [1973]). The potential economic value of this hormone sparked interest in obtaining BGH in commercial quantities at reasonable cost.
Thus, much work in recent years has focused on obtaining microbial synthesis of this commercially valuable hormone using recombinant DNA technology. Gene closing and manipulation techniques well known in the art have been used to produce recombinant expression vectors containing BGH-encoding cDNA fused to regulatory regions capable of directing synthesis of BGH in the desired host cells. Microorganisms transformed with these expression vectors have been shown to produce the desired hormone. For example, Keshet et al., (Nucleic Acids Research, 9:19-30 [1981]) reported the cloning and low level expression in E. coli of a full length BGH polypeptide as a fusion protein with a portion of pBR322-encoded .beta.-lactamase. In European Patent Application Publication No. 0 103 395, construction of several expression vectors, including vectors encoding BGH polypeptides with varying portions of the amino-terminal end deleted, is described. BGH polypeptides with varying portions of the amino-terminal end of the mature hormone deleted were found to retain biological activity and to be expressed at much higher levels than was the complete hormone in the expression systems described. Yields of BGH in various E. coli strains transformed with the expression vectors (and also with a plasmid carrying a gene encoding a temperature-sensitive repressor to control BGH synthesis) were 100 mg/liter or less in small-scale cultures. Large-scale fermentation of the transformed strains is not reported. Seeburg et al., (DNA, 2:37-45 [1983]) describe the cloning of bovine and porcine growth hormone cDNA and construction of expression vectors encoding the complete mature hormones (i.e., the "pre" or signal sequence region is removed in vitro during vector construction). E. coli cells were transformed with the BGH expression vector and BGH synthesis was regulated by the plasmid-borne E. coli trp regulatory region. It is reported that high density fermentation of the transformed E. coli cells yielded approximately 1.5 grams/liter BGH, but no description of the fermentation conditions is given.
Obtaining maximum expression levels of the protein products of cloned genes often involves some trial and error. The genes may be fused to several different regulatory regions and/or transformed into several host cell strains for comparative analyses to find the transformed strain giving the highest production levels of the desired protein. To date, efforts at yield improvement of microbially produced growth hormones have been carried out primarily at the level of genetic manipulations designed to increase cellular expression. There is still a need for the development of commercial scale fermentation processes capable of producing growth hormones in the highest possible yields.